I realize this may be a bit late but our mail port was not working
these messages were returned : D.G.
---------------------fm writes----------------------------------------
I recently posted the problems I had and appreciate your
responses. However, I am still not getting the mutants using
the Bio-rad kit. I am getting transformants with the negative
control DNA - where I make the second strand in the absense of
any mutagenic primer.
Just recently, I found that my SS DNA was able to transform XL1
blue competents. Apparently the SS DNA prep was contaminated
with DS DNA. Has anyone had experienced this problem? I am
making SS DNA from the Bluescript plasmid in CJ236 cells. I am
thinking of gel purifying the SS DNA. Is this a logical or a
stupid idea? I appreciate any comments.
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I have had similar problems with another strain and got rid of contaminating
DS DNA and RNA by treatment of supernatant with DNase I 10U/ml
and RNase A 10ug/mL for 15 min at 37 degrees C. Do this before PEG
pptting your phage and your ssDNA prep will be very clean!
Dan Gietz
University of Manitoba
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA